Materials and methods for mitigating immune-sensitization

ABSTRACT

The present invention concerns materials and method for delivery and internalization of an agent into a cell even in the presence of an immune response, such as antibodies or antisera or immune cells that bind to the agent. The agent can be a compound, drug, peptide, protein, nucleic acid, antigen, immunogen, or other biological molecule. In one embodiment, the agent is operatively linked to a lectin-based carrier. The present invention can be used for delivery and cellular internalization of any entity where an immune response to the entity is present or is likely to be produced or developed. The present invention also concerns methods and materials for providing for an adjuvant and carrier for vaccinations of a person or animal. The present invention also concerns a method for treating or preventing a disease or condition in a human or animal wherein the human or animal has produced or will produce an immune response against a therapeutic agent that can treat said disease or condition, the method comprising administering to the human or animal an effective amount of said therapeutic agent operatively linked to a lectin-based carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending U.S. patentapplication Ser. No. 16/079,932, filed Aug. 24, 2028; which is theNational Stage of International Application No. PCT/US2017/020235, filedMar. 1, 2017, which claims the benefit of U.S. Provisional ApplicationSer. No. 62/301,973, filed Mar. 1, 2016, and 62/456,443, filed Feb. 8,2017, each of which is hereby incorporated by reference herein in itsentirety, including any figures, tables, nucleic acid sequences, aminoacid sequences, or drawings.

The Sequence Listing for this application is labeled“SeqList-27Apr23.xml”, which was created on Apr. 27, 2023, and is 2 KB.The entire content is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Enzyme replacement therapies (ERTs) remain the most effective treatmentfor those rare genetic diseases for which approved recombinant enzymesare available. ERTs have been crucial in treating several lysosomalstorage diseases (LSD), which in their severe forms present withdevastating multi-organ pathologies in affected children. However, theinduction of patient antibodies (immune sensitization) directed againstthe therapeutic enzyme has emerged as a significant limitation in theeffectiveness of ERTs, altering enzyme distribution and activity.Reported rates of patients that develop significant immune sensitizationresponses following ERT are 91%, 97% and 100% for Hurler(Mucopolysaccharidosis I; MPS I), Maroteaux-Lamy (MPS VI), and Pompediseases, respectively (Dickson et al., 2008). Becauseearly/infantile-onset forms comprise the most severe LSD mutations(“nulls” with little/no detectable enzyme), the development of immunesensitization is much more prevalent in young patients. These childrencan show dramatic, often life-saving, improvements upon treatment onset.However, progress quickly declines as these children developneutralizing antibodies to the therapeutic enzyme.

The effectiveness of enzyme replacement therapies (ERT) for rare geneticdiseases and other peptide-, protein-, or glycoprotein-based therapiescan be undermined by the development of anti-drug antibodies (ADA), alsotermed immune-sensitization. All currently approved ERTs for LSDs, withthe exception of ERTs for Gaucher Disease, exploit themannose-6-phosphate (M6P) receptor for uptake into cells. Anti-ERTantibodies that interfere with M6P-mediated uptake constitute thepredominant “neutralizing” class of antibodies (Glaros et al., 2002).

BRIEF SUMMARY OF THE INVENTION

The present invention concerns materials and method for delivery andinternalization of an agent into a cell even in the presence of animmune response, such as antibodies or antisera, that binds to theagent. The agent can be a compound, drug, peptide, protein, nucleicacid, antigen, immunogen, or other biological molecule. In the method ofthe invention, the agent is operatively linked to a lectin-basedcarrier. The present invention can be used for delivery and cellularinternalization of any entity where an immune response to the entity ispresent or is likely to be produced or developed. The present inventionalso concerns methods and materials for providing for an adjuvant and/orcarrier for vaccinations or immunizations of a person or animal whereinimmune responses are induced to the antigen or immunogen but onlyminimally to the lectin-based carrier. The present invention alsoconcerns a method for treating a disease or condition in a human oranimal wherein the human or animal has produced or will produce animmune response against a therapeutic agent that can treat said diseaseor condition, the method comprising administering to the human or animalan effective amount of the therapeutic agent operatively linked to alectin-based carrier.

The invention exploits the unexpected low immunogenicity of thelectin-based carrier compared to the associated therapeutic and/orantigenic agent, which enables appropriate distribution and efficacy ofagent in individuals in which anti-agent immune responses reduce desiredtherapeutic or immunogenic responses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . IDUA:RTB uptake in presence of inhibitory levels of anti-IDUAantibodies. IDUA:RTB and mammalian cell-derived IDUA (rhIDU) werepre-incubated with canine sera from dogs no longer responsive to rhIDUtherapy (gift of P. Dickson, UCLA) were used to treat MSP I fibroblasts.Intracellular IDUA activity was analyzed at 1 and 4 h and compared tofibroblasts treated in the absence of neutralizing sera (Acosta, Cramerunpub.).

FIGS. 2A and 2B. ERT blocking antibodies. From Ponder, 2008 (Ponder,2008).

FIG. 3 . RTB trafficking.

FIG. 4 . RTB:IDUA corrects GAG levels +/−MMR and M6PR inhibitors. GAGlevels were measured in normal (GM00010) or MPS-I (GM01391) fibroblaststhat were untreated or treated for 24 hr with 1 unit IDUA eq./m1 usingRTB:IDUA or mcd-IDUA (mammalian cell-derived IDUA). For inhibitorstudies, MPS-I cells were pre-incubated for 2 hr with 4 mM M6P or 4mg/ml mannan prior to IDUA treatment (Acosta et al., 2016).

FIGS. 5A and 5B. The RTB carrier does not elicit antibody responses inmice receiving multiple administrations. MPS I mice were treated with 8weekly injections of IDUA:RTB at 0.58 mg/kg (human IDUA dose) or 2.0mg/kg. Sera, collected at 14, 35, and 63 days after initial treatment,was analyzed for presence of anti-IDUA (FIG. 5A) and anti-RTB (FIG. 5B)IgGs. Data presented is relative titer levels (anti-IgG ODs) at 63 dafter 8 treatments; n=8 for treated cohorts; n=6 for untreated mice.Both IDUA:RTB treatment groups showed significant mitigation of diseasesymptom.

FIG. 6 . Antibody isotyping in terminal serum after 8 weeklyadministration of 0.58 mg/Kg or 2.0 mg/Kg of IDUAL to MPS I−/− mice

FIG. 7 . GFP-specific serum IgG responses in ICR mice followingintranasal immunization with

-   -   1) 0.1 μg GFP    -   2) 0.1 μm GFP+0.1 μm cholera toxin (CT) adjuvant    -   3) 0.1 μm GFP+1 μg cholera toxin (CT)    -   4) 0.1 μm GFP+control non-transgenic hairy root media    -   5) 0.1 μm GFP as a RTB:GFP fusion purified from transgenic hairy        root media.    -   Groups of 5 mice were immunized, boosted on a 2-week schedule,        and bled 6 days after each boost. Titers were determined by        ELISA and were defined as the reciprocal of the highest dilution        of the serum giving an absorbance of ≥0.2 (three replicates per        determination). Each value is average+standard error of each        group. (Medina-Bolivar et al., 2003).

FIG. 8 . Adjuvant-specific serum IgG responses (anti-RTB or anti-CT) inmice trial described in FIG. 7 following nasal immunization with:

-   -   1) 0.1 μm GFP fused to 0.12 μg RTB    -   2) 0.1 μm GFP+0.1 μm CT    -   3) 0.1 μg GFP+1.0 μm CT.

FIG. 9 . Aim 2 overview and workflow.

FIG. 10 . A timeline for immunization and sample collection.

FIGS. 11A and 11B. IDUA:RTB treatment of MPS-I mice. FIG. 11A. IDUAactivity in untreated mice or mice 24 hr after IDUA:RTB injection. FIG.11B. GAG levels dpi. (Acosta et al., 2016; Ou et al., 2016).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of a modified patatin sequencethat can be used in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses the use of plant lectin-based deliveryof associated bioactive molecules (e.g., drugs) whereby thelectin-delivery-module enables efficacious drug delivery even in thepresence of anti-drug antibodies to the bioactive molecule. Thisinventions provides unique advantages in drug delivery in ADAimmune-sensitized patients independent of whether the elicitation ofimmunogenicity was initiated by

-   -   Multiple administrations of bioactive component such as an ERT        that lacks our inventive lectin carrier; i.e., patients        previously treated with a drug that subsequently developed ADA        that compromises drug treatment effectiveness    -   Treatment with the drug operatively associated with the        lectin-delivery-module resulting in antibodies raised to the        bioactive drug component (for example, as lectin-replacement        enzyme fusion)    -   Treatment by DNA- or RNA-based gene therapy approaches that        direct synthesis of the bioactive molecule in vivo leading to        ADA directed against the encoded therapeutic product.

These applications for patient treatments are based on the surprisingdiscovery that 1) the plant lectin B-subunit of ricin (RTB) is able tocarry its drug cargo into cells and direct appropriate subcellulartrafficking and transcytosis even in the presence of inhibitory levelsof neutralizing antibodies directed against its cargo, 2) the deliveredcargo shows bioactivity upon target-site delivery, and 3) the RTB lectincarrier itself shows surprisingly low immunogeniticy and does not sufferfrom reduced efficacy due to anti-carrier antibodies. The RTB carriermediates delivery and effective biodistribution of corrective ERT enzymein animals that are immune sensitized to a model ERT product. Theinvention provides for broad application of the lectin-delivery platformto treatments for lysosomal diseases, other genetic disorders, or drugdelivery in any case where immune sensitization to the drug isproblematic for therapeutic outcomes. Its utility is both for patientsfor whom their current treatment has declined in efficacy due to ADA andfor naive patients for initiating and maintaining treatment with theenzyme-lectin fusion providing long-term sustainable treatment that doesnot become compromised by ADA. Thus, the present invention brings newimmune-mitigating ERTs to patients that provide sustainable efficacy forERT treatments of lysosomal storage diseases and other genetic diseases.

One aspect of the present invention concerns materials and method fordelivery and internalization of an agent into a cell even in thepresence of an immune response, such as antibodies, antisera, and/orimmune cells (e.g., B cells, T cells, etc.), that binds to the agent. Inone embodiment, the antibodies, antisera, and/or immune cells areneutralizing antibodies, antisera, and/or immune cells. In the methodsof the invention, the agent is operatively linked to a lectin-basedcarrier (LBC). The agent can be any compound, drug, peptide, protein,nucleic acid, antigen, immunogen, or other synthetic or biologicalmolecule. In a specific embodiment, the lectin-based carrier is anon-toxic carbohydrate binding subunit of a plant lectin. In anexemplified embodiment, the plant lectin is the B-subunit of ricin(RTB), or a functional fragment or variant thereof. In specificembodiments, the ricin B subunit that is utilized is truncated byremoval of about 1 to 10 amino acids at the N-terminus of the protein.In a further embodiment, the ricin B subunit is truncated wherein thefirst six amino acids of the protein are removed. In another embodiment,the lectin is the nigrin B B-subunit (NBB) from Sambucus nigra, or afunctional fragment or variant thereof. In one embodiment, the agent isα-L-iduronidase, or an enzymatically active fragment or variant thereof.The present invention can be used for delivery and cellularinternalization of any entity where an immune response or immunecomponents to the entity are present or are likely to be produced ordeveloped.

The present invention also concerns a method for treating or preventinga disease or condition in a human or animal wherein the human or animalhas produced or will produce an immune response against a therapeuticagent that can treat said disease or condition, the method comprisingadministering to the human or animal an effective amount of thetherapeutic agent operatively linked to a lectin-based carrier. Theimmune response can be an antibody response and/or an immune cellresponse. In one embodiment, the antibody and/or immune cells areneutralizing antibody and/or immune cells. The agent can be anycompound, drug, peptide, protein, nucleic acid, antigen, immunogen, orother synthetic or biological molecule that is utilized in treating orpreventing a disease or condition. In a specific embodiment, thelectin-based carrier is a non-toxic carbohydrate binding subunit of aplant lectin. In a further embodiment, the plant lectin is the B-subunitof ricin (RTB), or a functional fragment or variant thereof. In specificembodiments, the ricin B subunit that is utilized is truncated byremoval of about 1 to 10 amino acids at the N-terminus of the protein.In an exemplified embodiment, the ricin B subunit is truncated whereinthe first six amino acids of the protein are removed. In anotherembodiment, the lectin is the nigrin B B-subunit (NBB) from Sambucusnigra, or a functional fragment or variant thereof.

The subject invention concerns materials and methods for the delivery oftherapeutic agents, such as drugs, peptides, proteins, antigens,immunogens, and polynucleotides, to a person or animal that has or willdevelop an immune response, such as an antibody response and/or immunecells response, against the therapeutic agent. In one embodiment, theantibody and/or immune cells are neutralizing antibody and/or immunecells. A method of the invention comprises administering a lectin thatcomprises a therapeutic agent to a person or animal in need of thetherapeutic agent. In one embodiment, the person or animal has alreadydeveloped antibodies against the therapeutic agent prior toadministration. In another embodiment, the person or animal is at riskof developing antibodies against the therapeutic agent. Any suitablelectin, such as a plant lectin, is contemplated for use in the method.In a specific embodiment, the lectin-based carrier is a non-toxiccarbohydrate binding subunit of a plant lectin. In a further embodiment,the plant lectin is the B-subunit of ricin (RTB), or a functionalfragment or variant thereof. In specific embodiments, the ricin Bsubunit that is utilized is truncated by removal of about 1 to 10 aminoacids at the N-terminus of the protein. In an exemplified embodiment,the ricin B subunit is truncated wherein the first six amino acids ofthe protein are removed. In another embodiment, the lectin is the nigrinB B-subunit (NBB) from Sambucus nigra, or a functional fragment orvariant thereof.

In some embodiments of the present invention, the therapeutic agent isfused or linked to the subunit B, or a fragment or variant thereof, ofan AB toxin. In some embodiments, the subunit B lectin protein is fromricin. In some embodiments, the ricin B subunit that is utilized istruncated by removal of about 1 to 10 amino acids at the N-terminus ofthe protein. In one embodiment, the ricin B subunit is truncated whereinthe first six amino acids of the protein are removed. A fusion protein(or other compound) may be produced by construction of a fusion geneincorporating a nucleotide sequence encoding a lectin (such as thesubunit B lectin) and a nucleotide sequence encoding the therapeuticprotein, and introducing this new genetic fusion (fusion gene) into aprotein expression system, expressing the fusion protein encoded by thefusion gene, and isolating the fused protein for use as a therapeuticdrug. Alternatively, the fusion may be accomplished by direct chemicalfusion or conjugation yielding fusion of the lectin (such as a subunit Bprotein) with the therapeutic agent. In one embodiment, the fusionprotein comprises a linker or spacer sequence of amino acids between thelectin and the therapeutic protein or compound. Examples of linker orspacer sequences are well known in the art. Methods for preparing fusiongenes and fusion protein are also well known in the art and have beendescribed, for example, in U.S. Pat. Nos. 7,964,377; 7,867,972;7,410,779; 7,011,972; 6,884,419; and 5,705,484.

The present invention also concerns methods and materials for providingfor an adjuvant and carrier for immunizations or vaccinations of aperson or animal wherein immune responses are induced to the antigen orimmunogen but only minimally to the lectin-based carrier. Thisadvantageously allows for multiple vaccinations and/or boosters, andreuse of the lectin-based carrier for multiple vaccine targets. In onemethod of the invention, a person or animal is administered an effectiveamount of an antigen or immunogen, wherein the antigen or immunogen isprovided operatively linked to a lectin-based carrier (LBC). Theantigen:LBC or immunogen:LBC is administered to a person or animal togenerate an immune response against the antigen or immunogen. In oneembodiment, the antigen:LBC or immunogen:LBC is administered to theperson or animal multiple times over a period of time. In a specificembodiment, the LBC is a non-toxic carbohydrate binding subunit of aplant lectin. In an exemplified embodiment, the plant lectin is theB-subunit of ricin (RTB), or a functional fragment or variant thereof.In specific embodiments, the ricin B subunit that is utilized istruncated by removal of about 1 to 10 amino acids at the N-terminus ofthe protein. In a further embodiment, the ricin B subunit is truncatedwherein the first six amino acids of the protein are removed. In anotherembodiment, the lectin is the nigrin B B-subunit (NBB) from Sambucusnigra, or a functional fragment or variant thereof. The presentinvention can be used as a vaccine delivery system for any knownimmunogen or vaccine or for any immunogen or vaccine developed in thefuture. In one embodiment, the immune response generated using thepresent invention comprises the production of antibodies that bind toone or more epitopes of the antigen or immunogen. In a furtherembodiment, the immune response generated is primarily a Th₂ response.In an exemplified embodiment, the antigen:LBC or immunogen:LBC isadministered at a mucosal location of the person or animal, e.g., nasaladministration. Compositions of the antigen:LBC or immunogen:LBC canoptionally comprise other adjuvants known in the art (e.g., alum,Freund's adjuvant, etc.) and/or physiologically-acceptable buffers, etc.

Plant lectins that are contemplated within the scope of the inventioninclude, but are not limited to those B subunits from AB toxins such asricins, abrins, nigrins, and mistletoe toxins, viscumin toxins, ebulins,pharatoxin, hurin, phasin, and pulchellin. They may also include lectinssuch as wheat germ agglutinin, peanut agglutinin, and tomato lectinthat, while not part of the AB toxin class, are still capable of bindingto animal cell surfaces and mediating endocytosis and transcytosis.Specific examples of plant lectins including their binding affinitiesand trafficking behavior are discussed further below. Therapeuticcompounds and agents contemplated within the scope of the inventioninclude, but are not limited to large molecular weight moleculesincluding therapeutic proteins and peptides, siRNA, antisenseoligonucleotides, and oligosaccharides. Other therapeutic compounds andagents contemplated within the scope of the invention include smallmolecular weight drug compounds including but not limited to vitamins,co-factors, effector molecules, and inducers of health promotingreactions.

Additional plant lectins that are contemplated within the scope of theinvention are those having particular carbohydrate binding affinitiesincluding but not limited to lectins that bind glucose, glucosamine,galactose, galactosamine, N-acetyl-glucosamine, N-acetyl-galactosamine,mannose, fucose, sialic acid, neuraminic acid, and/or N-acetylneuraminicacid, or have high affinity for certain target tissue or cells ofinterest. There are hundreds of plant lectins that have been identifiedand experimental strategies to identify plant lectins, their respectivegenes, and their sugar binding affinities are widely known by thoseskilled in the art. The diversity of plant sources for lectins and theirsugar binding affinities is exemplified in Table 1 below (adapted fromTable 3 of Van Damme et al., (1998)).

TABLE 1 Type 2 Ribosome inactivating Proteins and Related Lectins:Occurrence, Molecular Structure, and Specificity Sequence Species TissueStructure^(a) Specificity available^(b) Merolectins Sambucus nigra Bark[P22] NANA Nu Fruit [P22] NANA Nu Hololectins Sambutus nigra Bark II[P30]₂ GalNAc > Gal Nu Seed III [P30]₂ GalNAc > Gal Fruit IVf [P32]₂Gal/GalNAc Nu (SNA-IV) Leaf IVl [P32]₂ Gal/GalNAc Nu Leaf IV4l [P32]₄Gal/GalNAc Chimerolectins Abrus pracatonus Seed [P(34 + 32)] Gal >GalNAC Pr₄ Nu (Abrin) Seed [P(33 + 29)]₂ Gal Pr (APA) Ademia digitalaRoot [P(28 + 38)] Gal > GalNAc Adenia volkensii Root [P(29 + 36)] GalCinnamonum camphora Seed [P(30 + 33)]₂ Unknown Eranthis hyermalie Tuber[P(30 + 32)] GalNAc Iris hybrid Bulb [P(27 + 34)] GalNAc Momardicacharantis Seed [P(28 + 30)]₂ Gal > GalNAC Phoradendron califomicum Plant[P(31 + 38)] Gal Ricinus consurris Seed [P(32 + 34)] Gal > GalNAC Pr₄ Nu(Ririn) Seed [P(32 + 36)]₂ Gal > GalNAC Pr₄ Nu (RCA) Sambucus canadensisBark I [P(32 + 35)]₄ NANA Sambutus ebulus Bark I [P(32 + 37)]₄ NANA Leaf[P(26 + 30)]₃ GalNAc Sambuous nigra Seed Vs [P(26 + 32)]₃ GalNAc > GalBark I [P(32 + 35)]₄ NANA Nu (SNA-I) Bark I′ [P(32 + 36)]₃ NANA Nu(SNA-I′) Bark V [P(26 + 32)]₂ GalNAc > Gal Nu (SNA-V) Fruit If [P(32 +35)]₂ NANA Nu Fruit Vf [P(26 + 32)]₂ GalNAc > Gal Nu Sambucus racemosaBark I [P(30 + 36)]₄ NANA Sambucus sieboldiana Bark I [P(31 + 37)]₄ NANANu (SSA-I) Bark [P(27 + 32)] GalNAc > Gal Nu (Sieboldia) Viscum albumPlant I [P(29 + 34)]₁₋₂ gal Plant II [P(29 + 34)] Gal/GalNAc Plant III[P(25 + 30)] GalNAc > Gal Type 2 RIP with inactive B chain Sambutusnigra Bark [P(32 + 32)] . . . Nu (LRPSN) ^(a)[PX] stands for protomerwith a molecular mass of X kDa. [P(Y + Z)] indicates that the protomeris cleaved is two polypeptides of Y and Z kDA. ^(b)Pr₄ protein sequence;Nu, nucleotide sequence. The abbreviation in brackets refers to thesequence name used in the dendrogram (FIG. 20).

As a further example of plant lectins contemplated herein, Table 2 belowexemplifies the large number of different lectins identified from theSambucus species alone. This group includes nigrin B, the source on NBB.

TABLE 2 Ribosome-inactivating proteins (RIPs) and lectins from Sambucusspecies. Adapted from Table 1 of Ferreras et al. (2011) Proteins SpeciesTissues Type 1 RIPs Ebulitins α, β and γ S. ebulus Leaves Nigritins f1and f2 S. nigra Fruits Heterodimeric type 2 RIPs Ebulin 1 S. ebulusLeaves Ebulin f S. ebulus Fruits Ebulins r1 and r2 S. ebulus RhizomeNigrin b, basic nigrin b, SNA I′, SNLRPs S. nigra Bark Nigrins l1 and l2S. nigra Leaves Nigrin f S. nigra Fruits Nigrin s S. nigra SeedsSieboldin b S. sieboldiana Bark Basic racemosin b S. racemosa BarkTetrameric type 2 RIPs SEA S. ebulus Rhizome SNA I S. nigra Bark SNAIfS. nigra Fruits SNAflu-I S. nigra Flowers SSA S. sieboldiana Bark SRA S.racemosa Bark Monomeric lectins SELIm S. ebulus Leaves SEA II S. ebulusRhizome SNA II S. nigra Bark SNAIm and SNAIV1 S. nigra Leaves SNA IV S.nigra Fruits SNA III S. nigra Seeds SSA-b-3 and SSA-b-4 S. sieboldianaBark SRAbm S. racemosa Bark Homodimeric lectins SELld S. ebulus LeavesSELfd S. ebulus Fruits SNAld S. nigra Leaves

Any disease or disorder that can be treated or prevented using atherapeutic compound or agent is contemplated within the scope of thepresent invention. In one embodiment, the disease or disorder is one ofthe brain or CNS. Lysosomal diseases and (parenthetically) relatedenzymes and proteins associated with diseases that are contemplatedwithin the scope of the invention include, but are not limited to,Activator Deficiency/GM2 Gangliosidosis (beta-hexosaminidase),Alpha-mannosidosis (alpha-D-mannosidase), Aspartylglucosaminuria(aspartylglucosaminidase), Cholesteryl ester storage disease (lysosomalacid lipase), Chronic Hexosaminidase A Deficiency (hexosaminidase A),Cystinosis (cystinosin), Danon disease (LAMP2), Fabry disease(alpha-galactosidase A), Farber disease (ceramidase), Fucosidosis(alpha-L-fucosidase), Galactosialidosis (cathepsin A), Gaucher Disease(Type I, Type II, Type III) (beta-glucocerebrosidase), GM1gangliosidosis (Infantile, Late infantile/Juvenile, Adult/Chronic)(beta-galactosidase), I-Cell disease/Mucolipidosis II(GlcNAc-phosphotransferase), Infantile Free Sialic Acid StorageDisease/ISSD (sialin), Juvenile Hexosaminidase A Deficiency((hexosaminidase A), Krabbe disease (Infantile Onset, Late Onset)(galactocerebrosidase), Metachromatic Leukodystrophy (arylsulfatase A),Mucopolysaccharidoses disorders [Pseudo-Hurlerpolydystrophy/Mucolipidosis IIIA(N-acetylglucosamine-1-phosphotransferase), MPSI Hurler Syndrome(alpha-L iduronidase), MPSI Scheie Syndrome (alpha-L iduronidase), MPS IHurler-Scheie Syndrome (alpha-L iduronidase), MPS II Hunter syndrome(iduronate-2-sulfatase), Sanfilippo syndrome Type A/MPS III A (heparanN-sulfatase), Sanfilippo syndrome Type B/MPS III B(N-acetyl-alpha-D-glucosaminidase), Sanfilippo syndrome Type C/MPS III C(acetyl-CoA, alpha-glucosaminide acetyltransferase, Sanfilippo syndromeType D/MPS III D (N-acetylglucosamine-G-sulfate-sulfatase), Morquio TypeA/MPS IVA (N-acetylgalatosamine-6-sulfate-sulfatase), Morquio Type B/MPSIVB (β-galactosidase-1), MPS IX Hyaluronidase Deficiency(hyaluronidase), MPS VI Maroteaux-Lamy (arylsulfatase B), MPS VII SlySyndrome (beta-glucuronidase), Mucolipidosis I/Sialidosis(alpha-N-acetyl neuraminidase), Mucolipidosis IIIC(N-acetylglucosamine-1-phosphotransferase), Mucolipidosis type IV(mucolipin1)], Multiple sulfatase deficiency (multiple sulfataseenzymes), Niemann-Pick Disease (Type A, Type B, Type C)(sphingomyelinase), Neuronal Ceroid Lipofuscinoses [(CLN6disease—Atypical Late Infantile, Late Onset variant, Early Juvenile(ceroid-lipofuscinosis neuronal protein 6);Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease (battenin); FinnishVariant Late Infantile CLN5 (ceroid-lipofuscinosis neuronal protein 5);Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease(tripeptidyl peptidase 1); Kufs/Adult-onset NCL/CLN4 disease; NorthernEpilepsy/variant late infantile CLN8 (ceroid-lipofuscinosis neuronalprotein 8); Santavuori-Haltia/Infantile CLN1/PPT disease(palmitoyl-protein thioesterase 1); Beta-mannosidosis(beta-mannosidase)], Tangier disease (ATP-binding cassette transporterABCA1), Pompe disease/Glycogen storage disease type II (acid maltase),Pycnodysostosis (cathepsin K), Sandhoff disease/Adult Onset/GM2Gangliosidosis (beta-hexosaminidases A and B), Sandhoff disease/GM2gangliosidosis—Infantile, Sandhoff disease/GM2 gangliosidosis—Juvenile(beta-hexosaminidases A and B), Schindler disease(alpha-N-acetylgalactosaminidas), Salla disease/Sialic Acid StorageDisease (sialin), Tay-Sachs/GM2 gangliosidosis (beta-hexosaminidase),and Wolman disease (lysosomal acid lipase), Sphingolipidosis,Hurmansky-Pudiak Syndrome (HPS1, HPS3, HPS4, HPS5, HPS6 and HPS7) Type2—AP-3 complex subunit beta-1, Type 7—dysbindin), Chediak-HigashiSyndrome (lysosomal trafficking regulator protein), and Griscellidisease (Type 1: myosin-Va, Type 2: ras-related protein Rab-27A, Type 3:melanophilin).

Additional diseases (including related proteins) that may betherapeutically addressed by this invention include theneurodegenerative diseases which include but are not limited toParkinson's, Alzheimer's, Huntington's, and Amyotrophic LateralSclerosis ALS (superoxide dismutase), Hereditary emphysema(al-Antitrypsin), Oculocutaneus albinism (tyrosinase), Congenitalsucrase-isomaltase deficiency (Sucrase-isomaltase), and Choroideremia(Rep1) Lowe's Oculoceribro-renal syndrome (PIP2-5-phosphatase). Manyother genetic diseases are caused by deficiencies in specific proteinsor enzymes leading to disease specific tissue and organ pathologies.ERT's or other protein replacement therapeutics may be of value forthese diseases. Lectin-based carriers may facilitate protein delivery tocritical organs, cells and subcellular organelles or compartments forthese diseases as well.

An ERT delivery strategy has been developed that is based on the plantRTB lectin which mediates enzyme uptake and lysosomal trafficking byM6P-independent routes. This carrier supports enhanced biodistributionprofiles including the treatment of currently “hard-to-treat” tissuesand organs such as brain. The enzyme-RTB fusions can be produced using aplant-based bioproduction platform and thus do not contain M6P-modifiedglycans. The inventors have discovered that their enzyme-RTB fusionsprovide effective treatment even in conditions of immune-sensitizedhigh-titer antiserum with significant implications for patients in whichtreatment is undermined by high titer ADA.

The present invention contemplates products in which the lectin-basedcarrier is operatively associated with a therapeutic component,immunogen or antigen by one of many methods known in the art. Forexample, genetic fusions between a plant lectin protein and atherapeutic protein can orient the lectin partner on either the C- orN-terminus of the therapeutic component, immunogen or antigen. Thecoding regions can be linked precisely such that the last C-terminalresidue of one protein is adjacent to the first N-terminal residue ofthe mature (i.e., without signal peptide sequences) second protein.Alternatively, additional amino acid residues can be inserted betweenthe two proteins as a consequence of restriction enzyme sites used tofacilitate cloning at the DNA level. Additionally, the fusions can beconstructed to have amino acid linkers between the proteins to alter thephysical spacing. These linkers can be short or long, flexible (e.g.,the commonly used (Gly₄Ser)₃ ‘flexi’ linker) or rigid (e.g., containingspaced prolines), provide a cleavage domain (e.g., see Chen et al.(2010)), or provide cysteines to support disulfide bond formation. Theplant lectins are glycoproteins and in nature are directed through theplant endomembrane system during protein synthesis andpost-translational processing. For this reason, production ofrecombinant fusion proteins comprising a plant lectin and a therapeuticprotein partner may require that a signal peptide be present on theN-terminus of the fusion product (either on the lectin or on thetherapeutic protein depending on the orientation of the fusionconstruct) in order to direct the protein into the endoplasmic reticulumduring synthesis. This signal peptide can be of plant or animal originand is typically cleaved from the mature plant lectin or fusion proteinproduct during synthesis and processing in the plant or other eukaryoticcell. In one embodiment, a modified patatin signal sequence (PoSP) isutilized: MASSATTKSFLILFFMILATTSSTCAVD (SEQ ID NO:1) (see GenBankaccession number CAA27588.1, version GI:21514 by Bevan et al. andreferenced at “The structure and transcription start site of a majorpotato tuber protein gene” Nucleic Acid Res. 14 (11), 4625-4638 (1986)).

As used herein, compounds of the invention refers to the operativelylinked agent, immunogen, or antigen with the lectin-based carrier.Compounds of the subject invention can also be prepared by producing theplant lectin and the therapeutic agent, immunogen, or antigen separatelyand operatively linking them by a variety of chemical methods. Examplesof such in vitro operative associations include conjugation, covalentbinding, protein-protein interactions or the like (see, e.g., Lungwitzet al. (2005); Lovrinovic and Niemeyer (2005)). For example,N-hydroxysuccinimde (NHS)-derivatized small molecules and proteins canbe attached to recombinant plant lectins by covalent interactions withprimary amines (N-terminus and lysine residues). This chemistry can alsobe used with NHS-biotin to attach biotin molecules to the plant lectinsupporting subsequent association with streptavidin (which bindsstrongly to biotin) and which itself can be modified to carry additionalpayload(s). In another example, hydrazine-derivatized small molecules orproteins can be covalently bound to oxidized glycans present on theN-linked glycans of the plant lectin. Proteins can also be operativelylinked by bonding through intermolecular disulfide bond formationbetween a cysteine residue on the plant lectins and a cysteine residueon the selected therapeutic protein. It should be noted that the plantAB toxins typically have a single disulfide bond that forms between theA and B subunits. Recombinant production of plant B subunit lectins suchas RTB and NBB yield a product with an ‘unpaired’ cysteine residue thatis available for disulfide bonding with a “payload” protein.Alternatively, this cysteine (e.g., Cys₄ in RTB) can be eliminated inthe recombinant plant lectin product by replacement with a differentamino acid or elimination of the first 4-6 amino acids of the N-terminusto eliminate the potential for disulfide bonding with itself or otherproteins.

NBB: See GenBank accession number P33183.2, version GI:17433713(containing subunits A and B) by Van Damme et al. and referenced at“Characterization and molecular cloning of Sambucus nigra agglutinin V(nigrin b), a GalNAc-specific type-2 ribosome-inactivating protein fromthe bark of elderberry (Sambucus nigra)” Eur. J. Biochem. 237 (2),505-513 (1996). PDB ID: 3CA3 (for B subunit) by Maveyraud et al. andreferenced at “Structural basis for sugar recognition, including the tocarcinoma antigen, by the lectin sna-ii from sambucus nigra” Proteins 75p.89 (2009).

RTB: See GenBank accession number pbd/2AAI/B, version GI:494727(containing subunits A and B) by Montfort et al. and referenced at “Thethree-dimensional structure of ricin at 2.8A” J. Biol Chem. 262 (11),5398-5403 (1987).

In vivo administration of the subject compounds (i.e., an agentoperatively linked to an LBC) and compositions containing them, can beaccomplished by any suitable method and technique presently orprospectively known to those skilled in the art. The subject compoundscan be formulated in a physiologically- or pharmaceutically-acceptableform and administered by any suitable route known in the art including,for example, oral, nasal, rectal, transdermal, vaginal, and parenteralroutes of administration. As used herein, the term parenteral includessubcutaneous, intradermal, intravenous, intramuscular, intraperitoneal,and intrasternal administration, such as by injection. Administration ofthe subject compounds of the invention can be a single administration,or at continuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compounds of the subject invention, and compositions comprisingthem, can also be administered utilizing liposome and nano-technology,slow release capsules, implantable pumps, and biodegradable containers,and orally or intestinally administered intact plant cells expressingthe therapeutic product. These delivery methods can, advantageously,provide a uniform dosage over an extended period of time.

Compounds of the subject invention (i.e., an agent operatively linked toan LBC) can be formulated according to known methods for preparingphysiologically acceptable compositions. Formulations are described indetail in a number of sources which are well known and readily availableto those skilled in the art. For example, Remington's PharmaceuticalScience by E. W. Martin describes formulations which can be used inconnection with the subject invention. In general, the compositions ofthe subject invention will be formulated such that an effective amountof the compound is combined with a suitable carrier in order tofacilitate effective administration of the composition. The compositionsused in the present methods can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional physiologically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the subject compounds include ethanol, dimethyl sulfoxide,glycerol, alumina, starch, saline, and equivalent carriers and diluents.To provide for the administration of such dosages for the desiredtherapeutic treatment, compositions of the invention will advantageouslycomprise between about 0.1% and 99%, and especially, 1 and 15% by weightof the total of one or more of the subject compounds based on the weightof the total composition including carrier or diluent.

Compounds of the invention, and compositions thereof, may be locallyadministered at one or more anatomical sites, optionally in combinationwith a pharmaceutically acceptable carrier such as an inert diluent.Compounds of the invention, and compositions thereof, may besystemically administered, such as intravenously or orally, optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent, or an assimilable edible carrier for oral delivery. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

Compounds and compositions of the invention, including pharmaceuticallyacceptable salts or analogs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compound ofthe invention in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Useful dosages of the compounds and pharmaceutical compositions of thepresent invention can be determined by comparing their in vitroactivity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

The present invention also concerns pharmaceutical compositionscomprising a compound of the invention in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, comprising an amount ofa compound constitute a preferred embodiment of the invention. The doseadministered to a patient, particularly a human, in the context of thepresent invention should be sufficient to achieve a therapeutic responsein the patient over a reasonable time frame, without lethal toxicity,and preferably causing no more than an acceptable level of side effectsor morbidity. One skilled in the art will recognize that dosage willdepend upon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsof the invention can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds based on the weight of the total composition including carrieror diluents. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

The subject invention also concerns kits comprising a compound and/orcomposition of the invention in one or more containers. Kits of theinvention can optionally include pharmaceutically acceptable carriersand/or diluents. In one embodiment, a kit of the invention includes oneor more other components, adjuncts, or adjuvants as described herein. Inone embodiment, a kit of the invention includes instructions orpackaging materials that describe how to administer a compound orcomposition of the kit. Containers of the kit can be of any suitablematerial, e.g., glass, plastic, metal, etc., and of any suitable size,shape, or configuration. In one embodiment, a compound of the inventionis provided in the kit as a solid, such as a tablet, pill, or powderform. In another embodiment, a compound of the invention is provided inthe kit as a liquid or solution. In one embodiment, the kit comprises anampoule or syringe containing a compound of the invention in liquid orsolution form.

Mammalian species which benefit from the disclosed methods include, butare not limited to, primates, such as apes, chimpanzees, orangutans,humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats,guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, andferrets; domesticated farm animals such as cows, buffalo, bison, horses,donkey, swine, sheep, and goats; exotic animals typically found in zoos,such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,seals, sea lions, elephant seals, otters, porpoises, dolphins, andwhales. Other species that may benefit from the disclosed methodsinclude fish, amphibians, avians, and reptiles. As used herein, theterms “patient” and “subject” are used interchangeably and are intendedto include such human and non-human species. Likewise, in vitro methodsof the present invention can be carried out on cultured cells or tissuesof such human and non-human species.

As used herein, the terms “nucleic acid” and “polynucleotide” refer to adeoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide andribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, would encompass known analogs of naturalnucleotides that can function in a similar manner as naturally-occurringnucleotides. The polynucleotide sequences include the DNA strandsequence that is transcribed into RNA and the strand sequence that iscomplementary to the DNA strand that is transcribed. The polynucleotidesequences also include both full-length sequences as well as shortersequences derived from the full-length sequences. Allelic variations ofthe exemplified sequences also fall within the scope of the subjectinvention. The polynucleotide sequence includes both the sense andantisense strands either as individual strands or in the duplex.

Techniques for transforming plant cells with a polynucleotide or geneare known in the art and include, for example, Agrobacterium infection,viral vectors, transient uptake and gene expression in plant seedlings,biolistic methods, electroporation, calcium chloride treatment,PEG-mediated transformation, etc. U.S. Pat. No. 5,661,017 teachesmethods and materials for transforming an algal cell with a heterologouspolynucleotide. Transformed cells can be selected, redifferentiated, andgrown into plants that contain and express a polynucleotide of theinvention using standard methods known in the art. The seeds and otherplant tissue and progeny of any transformed or transgenic plant cells orplants of the invention are also included within the scope of thepresent invention. Likewise, techniques for expressing recombinantproteins in other eukaryotic production systems that include but are notlimited to yeast, insect cell/baculovirus systems, mammalian cells, ortransgenic animals is well known in the art.

Because of the degeneracy of the genetic code, a variety of differentpolynucleotide sequences can encode polypeptides and enzymes of thepresent invention. A table showing all possible triplet codons (andwhere U also stands for T) and the amino acid encoded by each codon isdescribed in Lewin (1985). In addition, it is well within the skill of aperson trained in the art to create alternative polynucleotide sequencesencoding the same, or essentially the same, polypeptides and enzymes ofthe subject invention. These variant or alternative polynucleotidesequences are within the scope of the subject invention. As used herein,references to “essentially the same” sequence refers to sequences whichencode amino acid substitutions, deletions, additions, or insertionswhich do not materially alter the functional activity of the polypeptideencoded by the polynucleotides of the present invention. Allelicvariants of the nucleotide sequences encoding a wild type polypeptide ofthe invention are also encompassed within the scope of the invention.

Substitution of amino acids other than those specifically exemplified ornaturally present in a wild type polypeptide or enzyme of the inventionare also contemplated within the scope of the present invention. Forexample, non-natural amino acids can be substituted for the amino acidsof a polypeptide, so long as the polypeptide having the substitutedamino acids retains substantially the same biological or functionalactivity (e.g., enzymatic, or binding capability of a lectin) as thepolypeptide in which amino acids have not been substituted. Examples ofnon-natural amino acids include, but are not limited to, ornithine,citrulline, hydroxyproline, homoserine, phenylglycine, taurine,iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, α-aminohexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline,cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, C-methyl amino acids, N-methyl aminoacids, and amino acid analogues in general. Non-natural amino acids alsoinclude amino acids having derivatized side groups. Furthermore, any ofthe amino acids in the protein can be of the D (dextrorotary) form or L(levorotary) form. Allelic variants of a protein sequence of a wild typepolypeptide or enzyme of the present invention are also encompassedwithin the scope of the invention.

Amino acids can be generally categorized in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a polypeptide or enzyme of the present inventionhaving an amino acid of one class is replaced with another amino acid ofthe same class fall within the scope of the subject invention so long asthe polypeptide having the substitution still retains substantially thesame biological or functional activity (e.g., enzymatic, or bindingcapability of a lectin) as the polypeptide that does not have thesubstitution. Polynucleotides encoding a polypeptide or enzyme havingone or more amino acid substitutions in the sequence are contemplatedwithin the scope of the present invention. Table 3 provides a listing ofexamples of amino acids belonging to each class.

TABLE 3 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

The subject invention also concerns variants of the polynucleotides ofthe present invention that encode functional or biologically activepolypeptides of the invention. Variant sequences include those sequenceswherein one or more nucleotides of the sequence have been substituted,deleted, and/or inserted. The nucleotides that can be substituted fornatural nucleotides of DNA have a base moiety that can include, but isnot limited to, inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine,1-methylguanine, 5-methylcytosine, and tritylated bases. The sugarmoiety of the nucleotide in a sequence can also be modified andincludes, but is not limited to, arabinose, xylulose, and hexose. Inaddition, the adenine, cytosine, guanine, thymine, and uracil bases ofthe nucleotides can be modified with acetyl, methyl, and/or thio groups.Sequences containing nucleotide substitutions, deletions, and/orinsertions can be prepared and tested using standard techniques known inthe art.

Fragments and variants of a polypeptide or enzyme of the presentinvention can be generated as described herein and tested for thepresence of biological (e.g., binding capability) or enzymatic functionusing standard techniques known in the art. Thus, an ordinarily skilledartisan can readily prepare and test fragments and variants of apolypeptide or enzyme of the invention and determine whether thefragment or variant retains functional or biological activity (e.g.,enzymatic activity) relative to full-length or a non-variantpolypeptide.

Polynucleotides and polypeptides contemplated within the scope of thesubject invention can also be defined in terms of more particularidentity and/or similarity ranges with those sequences of the inventionspecifically exemplified herein or known in the art. The sequenceidentity will typically be greater than 60%, preferably greater than75%, more preferably greater than 80%, even more preferably greater than90%, and can be greater than 95%. The identity and/or similarity of asequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99% as compared to a sequence exemplified herein. Unless otherwisespecified, as used herein percent sequence identity and/or similarity oftwo sequences can be determined using the algorithm of Karlin andAltschul (1990), modified as in Karlin and Altschul (1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (1990). BLAST searches can be performed with the NBLASTprogram, score=100, wordlength=12, to obtain sequences with the desiredpercent sequence identity. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be used as described in Altschul et al.(1997). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (NBLAST and XBLAST) can be used.See NCBI/NIH website.

Single letter amino acid abbreviations are defined in Table 4.

TABLE 4 Letter Symbol Amino Acid A Alanine B Asparagine or aspartic acidC Cysteine D Aspartic Acid E Glutamic Acid F Phenylalanine G Glycine HHistidine I Isoleucine K Lysine L Leucine M Methionine N Asparagine PProline O Glutamine R Arginine S Serine T Threonine V Valine WTryptophan Y Tyrosine Z Glutamine or glutamic acid

Hurler (the most severe form of MPS I) has been used extensively todelineate ERT immune sensitization and tolerance mechanisms withwell-characterized mouse, rat and canine disease models. Recombinantprotein comprising human α-L-iduronidase fused to the RTB lectin(IDUA:RTB) was produced and tested for uptake into cultured human MPS Ipatient fibroblast cells in the presence of canine serum (gift of Dr.Patricia Dickson, UCLA) containing high-titer neutralizing antibody tomammalian cell-derived iduronidase (rhIDU). Our IDUA:RTB showed greaterthan 90% efficiency in enzyme delivery in the presence of inhibitorycanine serum, in contrast to <10% uptake of mammalian cell-derived rhIDUcontaining M6P FIG. 1 (discussed further below). Animal disease modelsare similarly used to show that the RTB lectin fusion drugs mitigateimmune-sensitization issues in vivo.

Lysosomal Storage Diseases as a model for ADA treatment limitations.Lysosomal Storage Disorders (LSD) are a group of rare genetic diseasesin which a defect in a lysosomal hydrolase or other protein affectslysosomal function, resulting in accumulation of specific storageproducts in cell and tissues. LSDs have an estimated combined incidenceof 1 in 7,000-8,000 live births. Enzyme replacement therapies (ERTs) arethe treatment of choice and currently six LSDs have approved ERTs. TheseERTs have been very beneficial for patient care and quality of life.However, ERTs are often compromised by immune responses—the developmentof neutralizing antibodies to the therapeutic enzyme. Antibody responseto the ERT has been reported to hinder efficacy of the treatment in fiveout of the six ERTs available in patients and is prevalent in animalmodels used for R&D of new ERTs [3,4]. Over 90% of the patients withHurler (MPS I), MPS VI, Pompe and Fabry develop antibody to the drug(Wang et al., 2008). Infants and children are most affected, as patientswith early onset forms typically have no residual enzyme (i.e., arecross reactive immunological material negative; CRIM⁻) and quicklydevelop neutralizing antibodies that abrogate therapeutic correction andcan precipitate severe adverse clinical effects. Impacts on Pompepatients are often cited as the most dramatic example of thesereactions, where children show significant improvement in motormilestones in response to ERT only to have these effects rapidlyreversed upon induction of neutralizing antibodies, leading eventuallyto patient death (Wang et al., 2008; Nayak et al., 2014; Kishnani etal., 2010). Tolerization protocols have been tested for patients whereERT efficacy is blocked by immune responses, but these treatments arevery challenging and intensive for patients and pose significant risksof infection or malignancy (Wang et al., 2008). Gene therapy approachesare currently under development for numerous LSDs as an alternative toERTs. However, therapeutic enzymes produced via gene therapy also leadto neutralizing antibody responses in CRIM⁻ patients and animals (Xu etal., 2004) underscoring the need to develop new approaches to addressthe challenges imposed by immune responses to therapeutic proteins. Theinventors' novel lectin-based cell-targeting carrier for therapeuticenzymes, based on fusion to the plant lectin RTB, has been shown toretain full corrective efficacy in vitro in the presence of neutralizingantibodies directed against the enzyme. Studies in MPS I patients andanimals have contributed greatly to the understanding of antibodyresponse effects and mechanisms, and provide well characterized toolsand animal models to study these complex interactions.

Mucopolysaccharidosis I—ERT and immune sensitization. MPS I, also calledHurler, Hurler/Scheie, or Scheie Syndrome depending on disease severity,is a chronic, progressive lysosomal storage disorder. It is caused bydeficiencies in α-L-iduronidase (IDUA) resulting in pathogenicaccumulation of glycosaminoglycans (GAG) in lysosomes throughout thebody. Failure to effectively clear GAG leads to clinical manifestationsaffecting the heart, bones and joints, organs of the viscera, eyes,respiratory system, facial features, and the CNS. In its most severeform (Hurlers Syndrome), symptoms are evident in infancy leading toearly death (median age 6.8 years). Current MPS I treatment options areprimarily enzyme replacement therapy and/or hematopoietic stem celltransplantation. Recombinant human IDUA (rhIDU; ALDURAZYME®) produced inmammalian cells is currently available to MPS I patients and long-termERT treatment has proven effective in reducing many of the visceralmanifestations of the disease although the CNS, corneal clouding, andbone defects prevalent in MPS I are not improved. Cell uptake andlysosomal delivery of ALDURAZYME® is based on the interaction of IDUAwith mannose-6-phosphate receptors (M6PR) on target cells.

Among MPS I patients receiving ALDURAZYME®, 91% develop anti-rhIDU IgGantibodies. Evidence of reduced drug efficacy includes elevated urinaryGAG excretion in patients with high antibody titers to rhIDU (Wraith etal., 2007). In animal models, development of anti-rhIDU antibodiesalters the biodistribution in organs and organelles (Turner et al.,2000) and reduces delivery of enzyme to organs by interfering with theM6P receptor-mediated uptake mechanism (Dickson et al., 2008; Glaros etal., 2002; Ponder, 2008) (FIGS. 2A and 2B).

Lectin-based carriers provide new mechanisms of ERT uptake and lysosomaltrafficking. We have developed the RTB plant lectin as a novel carrierfor lysosomal enzymes. RTB is the non-toxic carbo-hydrate-binding Bsubunit of the plant type II AB toxin, ricin. RTB facilitates uptake bytargeting cell surface glycoproteins and glycolipids with (3-1,4-linkedgalactose or galactosamine residues. These are abundant on mammaliancells (Sandvig et al., 2014; Olsnes, 2004), providing access to a broadarray of cells. RTB enters cells by at least 6 different endocytoticroutes including both absorptive- and receptor-mediated mechanisms(Sandvig et al., 2011; Sandvig et al., 1999; Simmons et al., 1986;Frankel et al., 1997). Upon endocytosis, RTB traverses preferentially tolysosomes (FIG. 3 ) or cycles back to the cell membrane (transcytosispathway), with less than 5% moving “retrograde” to the endoplasmicreticulum (route for RTA toxin delivery) (Olsnes, 2004; Van Deurs etal., 1986). RTB fusions (both RTB:IDUA and IDUA:RTB) were produced usinga transient plant-based expression system.

Lectin-IDUA fusions corrects GAG levels by M6P receptor-independentmechanisms. Plant-made RTB:IDUA and IDUA:RTB fusion proteins retain bothRTB lectin binding activity and IDUA enzyme activity. Unlike mammaliancells, plant cells do not possess the enzymatic machinery to makeM6P-modified glycans (He et al., 2013). Thus, mammalian cell uptake ofthese fusion products is solely mediated by the RTB lectin. Todemonstrate this, purified RTB:IDUA product was used to treat MPSI/Hurler patient fibroblasts. Treatment with the RTB fusion productresulted in GAG reduction to “normal” levels comparable to controlmammalian cell-derived rhIDU. As shown in FIG. 4 , RTB-mediated deliveryof IDUA was independent of mannose-6-phosphate receptors (in contrast torhIDU) and high-mannose receptors (Acosta et al., 2015). Our in vitrodata indicate that RTB efficiently delivers functional enzyme into cellsand mediates disease substrate clearance by mechanisms that arefundamentally different than all current ERTs for LSDs. In vivo data inMPS I mice indicate biodistribution of IDUA:RTB to multiple visceralorgans following a single tail-vein injection and disease correctionbased on GAG levels, loss of splenomegaly, and improved memory/learningfollowing multiple weekly injections (Acosta et al., 2016; Ou et al.,2016). Thus, IDUA:RTB functions as a highly effective ERT.

Plant-based bioproduction. Our IDUA:RTB fusion is produced in a rapidplant-based transient expression system (Whaley et al., 2011; Komarovaet al., 2010). Plants provide advantages in safety (no adventitiousviral contamination issues) and cost of manufacture and have been shownto synthesize fully functional human lysosomal enzymes (U.S. Pat. No.5,929,304). In 2012, FDA-approved plant-derived Elelyso(Protalix/Pfizer) for treatment of Gaucher patients. Elelyso is beingoffered at 75% of the cost of the CHO-derived product. This product hasbeen administered to patients for 8 years (Aviezer et al., 2009) andshows no increase in immunogenicity compared to mammalian cell-derivedglucocerebrosidase (Grabowski et al., 2014) and is well tolerated bypatients switching from animal-cell-derived products (Grabowski et al.,2014; Pastores et al., 2013).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1

Patients develop immune responses to protein-based treatments that canlead to reduced efficacy (or abolishment of efficacy), alteredbiodistribution, and, in some cases, life-threatening adverse immuneresponses. BioStrategies has been developing the lectin component(non-toxic carbohydrate-binging B-subunit) of plant AB toxins ascarriers for associated human proteins. These include human lysosomalenzymes capable of treating lysosomal diseases by providing the proteinsthat are genetically deficient termed “enzyme replacement therapies”(ERTs). Our lead plant lectin is RTB (B-subunit of ricin). RTB binds togalactose and galactosamine residues that are abundant on the surface ofhuman cells, triggers endocytosis, and directs trafficking of associatedproteins using the endosome to lysosome and transcytosis pathways. Wehave produced a variety of RTB fusion proteins linking RTB to enzymesand other proteins using a plant-based protein bioproduction platform.We have demonstrated that RTB effectively delivers the associated“payload” proteins into mammalian cells both in vitro and in multiple invivo mouse disease models. Once bound to the cell surface, RTB triggersadsorptive-mediated endocytosis and transcytosis and has been shown tosupport broad in vivo biodistribution including cells of so-called“hard-to-treat” tissues and organs (e.g., brain, heart, lung). Analysesin mice and in human cells have revealed two unexpected features of theplant lectin carrier that suggests highly unique and useful interactionswith the immune system. 1) Although serum antibodies were developedagainst the “payload” protein (two examples—human iduronidase asIDUA:RTB fusion and the green fluorescent protein as RTB:GFP) followingmultiple administrations in mice, no antibodies directed against the RTBlectin were detected (see FIGS. 5A and 5B). And 2) if the IDUA:RTBprotein was exposed to neutralizing antibodies to the IDUA component(from animals that had lost the ability to respond to corrective enzymedue to the high-titer of neutralizing anti-IDUA antibodies in theirserum) and then used to treat disease cells, the RTB carrier was stillable to support cell uptake and lysosomal delivery, and provide activeenzyme that degraded the disease substrate to correct the lysosomaldisease cellular phenotype.

Thus plant lectins provide novel and unanticipated advantages as“stealth carriers” for associated therapeutic macromolecules that mayelicit anti-drug immunogenicity. These advantages include:

-   -   Enzymes fused with RTB will restore efficacy in patients that        have already developed neutralizing anti-ERT or anti-drug immune        responses that undermine treatment efficacy; and    -   Patients initiated with treatment comprising RTB fused to a        therapeutic component will retain treatment efficacy even if        serum antibodies are developed to the therapeutic macromolecule;        and    -   Patients can be treated with additional therapeutic agents that        employ the RTB carrier providing similar distribution and        trafficking patterns that would not be disrupted by potential        anti-carrier antibodies.

Potential product immunogenicity. Development of anti-drug immuneresponses has been challenging for many LSD patients receiving currentlyapproved ERT biologics. BioStrategies has initiated several studies toassess immune impacts of our RTB:enzyme fusions. Initial resultsindicate that RTB not only lacks significant immunogenicity itself, butalso may provide advantages as an enzyme carrier in patients withinhibitory antibodies to their current ERT drug.

We demonstrate here in FIG. 1 that RTB delivers corrective ERT dosesinto cells even in the presence of inhibitory levels of anti-ERTneutralizing antibodies. In vivo results (FIGS. 5A and 5B) on inductionof serum antibodies in MPS I (IDUA^(−/−)) mice following 8 weeklyinjections of plant-made IDUA:RTB (Acosta et al., 2016; Ou et al., 2016)have also been performed. These data indicate that MPS I mice developanti-IDUA antibody titers analogous to that seen following treatmentwith mammalian cell derived IDUA (Ou et al., 2014; Baldo et al., 2013).In contrast, mice did not develop antibodies directed against RTB (seeFIG. 5B) or against the glycans of plant-made IDUA.

Humoral response was further analyzed by isotyping the antibodiespresent in the terminal serum. More than 99% of the immunoglobulinsproduced belongs to the IgG1 subclass, a typical response againstprotein and peptide antigens. Insignificant production of other isotypesubclasses suggests that the response is not triggered by thecarbohydrates/polysaccharides present in the protein (IgG2, IgA) or dueto an allergic reaction against the therapeutic protein (IgE) FIG. 6 .

EXAMPLE 2

Mucosal vaccines, those delivered intranasally or orally, can be highlyeffective in triggering both systemic and mucosal immune responses.However, of the 22 vaccines currently in routine use for non-biowarfareinfectious diseases, 20 are delivered by injection and stimulate onlysystemic immunity. Although potential “protective antigens” have beenidentified for many disease agents including Category A and B agents,they generally require an “adjuvant” or specific carrier in order totrigger a strong immune response. For injectable vaccines, alum(aluminum hydroxide; an irritant) and more recently, MF59 (a mix ofsqualene and surfactants) are the only adjuvants currently approved bythe FDA for use in humans. However, these compounds are not effectiveadjuvants for mucosal vaccines. Cholera toxin has been the “goldstandard” mucosal adjuvant for nasal and oral delivery of vaccines inrodents but is not approved for humans because of associated toxicity.We have developed the non-toxic carbohydrate-binding subunit of ricintoxin (B subunit; RTB) as a mucosal adjuvant and carrier anddemonstrated that RTB provides vaccine adjuvancy equivalent to choleratoxin for nasal delivery (Medina-Bolivar et al., 2003). Thisgalactose/galactosamine-binding lectin binds to human mucosal surfaces(including a high affinity for M-cells), and thus functions to deliverfused antigens directly to immune-responsive cells of the mucosa. Theefficacy of RTB as an antigen delivery system for mucosal vaccines wasdemonstrated using the green fluorescent protein (GFP) as a modelantigen. GFP was genetically fused to RTB and expressed in tobaccoplants and in root cultures derived from these plants (Medina-Bolivar etal., 2003). Tobacco-synthesized RTB:GFP, a 62 kD glycoprotein whichretains both GFP fluorescence and RTB carbohydrate binding specificity,was affinity-purified from the media of root cultures using agalactosamine resin and used for nasal immunization of mice. The immuneresponses of mice immunized intranasally with GFP alone, GFP pluscholera toxin adjuvant, or affinity-purified RTB:GFP from tobacco werecompared. As shown in FIG. 7 , RTB:GFP triggered significant increasesin GFP-specific serum IgGs. This strong humoral response was comparableto that observed following GFP immunization with cholera toxin adjuvant.GFP at the same concentrations but without an adjuvant wasnon-immunogenic. Induction of higher levels of IgG₁ than IgG_(2a)following RTB:GFP immunization suggested that RTB, like CT, mediatesprimarily a Th2 response. Serum and fecal anti-GFP IgAs were alsoelevated at levels equivalent to that seen with cholera toxin asadjuvant (Medina-Bolivar et al., 2003), supporting the effectiveness ofRTB as an adjuvant and antigen carrier to the mucosa.

RTB functions as a “stealth” adjuvant. Most protein-based mucosaladjuvants (e.g., CT, LT, CT/LT derivatives, mistletoe lectin,proteosomes) are strongly immunogenic eliciting high serum titers ofanti-adjuvant antibodies. This has raised concerns of reduced adjuvancyin later boosts or as a component of a distinct vaccine. Morecritically, concerns have been raised as to whether reuse of theseadjuvants in a subsequent vaccine might trigger adverse hypersensitiveimmune responses. In preliminary studies, RTB shows striking differencesin its intrinsic immunogenicity compared to CT (and reports of LT andmistletoe lectin adjuvants). As shown in FIG. 8 , antibodies specific toRTB were not detected in serum of mice immunized intranasally withRTB:GFP even though high titers of anti-GFP antibodies were induced (seeFIG. 7 ). In contrast, high levels of anti-CT antibodies were present inmice immunized with CT+GFP (FIG. 8 ). This is not due solely to the lowlevel of RTB used (100 ng/dose) since CT at 100 ng/dose was highlyimmunogenic. Thus, RTB may be unusually non-immunogenic as an adjuvant.In support of this, several groups have tried to use RTB as a protectiveantigen to develop vaccines against ricin toxin (RTA+RTB) withoutsuccess (N. Mantis, Children's Hosp. Boston; G. Glenn, Walter Reed ArmyInst. of Research, pers. comm.). To further delineate this intriguingfeature of RTB and expand its utility as an adjuvant and mucosalcarrier, experiments are designed to assess the levels of anti-RTB IgGsfollowing mucosal immunizations with higher doses of RTB:GFP fusion,additional boosts extended over longer periods, and followingadministration of a second antigen-RTB fusion. This “stealth”characteristic of RTB has significant implications with respect toregulatory acceptance and “reuse” potential in multiple vaccines. Forexample, RTB could serve as carrier for a subunit Ebola antigen vaccinewith protective immunity elicited by multiple vaccination/boostprotocols to gain robust protection against the Ebola virus. The samepatient could be immunized later with a vaccine for protection againstZika virus using a Zika antigen associated with the RTB carrier. SinceRTB itself is non-immunogenic, there would be no immune suppressioncaused by the previous exposure to RTB that could undermine the desiredimmune response to the Zika antigen.

EXAMPLE 3

RTB delivers IDUA in presence of inhibitory anti-rhIDU antibodies.Neutralizing canine serum from rhIDU-immunized animals inhibits ERTuptake in human MPS I fibroblast by interfering with the M6P receptors(Dickson et al., 2008). To determine if RTB will deliver correctivedoses of human IDUA into disease cells in the presence of neutralizingantibodies, we compared cell uptake of IDUA:RTB versus rhIDU followingpre-incubation with neutralizing canine serum (provided by P. Dickson).Consistent with previous reports (Dickson et al., 2008), serum from highanti-rhIDU titer dogs inhibited uptake of mammalian cell-derived rhIDUby greater than 90% (FIG. 1 ). In contrast, IDUA:RTB showed 70%inhibition at 1 hr but by 4 hr, 90% of the IDUA:RTB was successfullytaken up into cells (FIG. 5 ). These results suggest that RTB deliverytechnology will facilitate delivering of corrective enzyme in patientsthat have developed uptake-blocking antibodies to rhIDU. This should bebroadly applicable to other disease treatments where ADA impactscellular uptake and trafficking of drug.

EXAMPLE 4

The RTB carrier module is itself non-immunogenic. In one example,wildtype mice were transnasally vaccinated and boosted 2-3 times withRTB:GFP fusions as described in (Medina-Bolivar et al., 2003). They werevaccinated in the presence of Freund's adjuvant in order to elicitstrong immunity. Although all vaccinated mice developed strong antibodytiters to the GFP “cargo”, essentially no antibodies above backgroundwere detected against RTB. In two other examples, knockout mice for twodifferent lysosomal diseases were treated with lysosomal enzyme:RTBfusions for 4 to 6 weeks at various doses in trials that demonstrateddrug efficacy in disease correction. Serum was collected at multipletimes and tested for induction of anti-drug antibodies. Antibodies(IgGs) against the human lysosomal enzyme component were detected withlevels comparable to those reported in the literature when mice aretreated with the mammalian-cell-derived enzyme alone (i.e., no RTB). Incontrast, anti-RTB titers in the same animals were very low (essentiallybackground).

These data indicate that although RTB carries an immunogenic proteincargo, the carrier itself shows very low immunogenicity—is a “stealth”carrier. Because RTB is capable of directing cell uptake even when thecargo is bound by serum antibodies (see FIG. 1 ), RTB-Enzyme fusionsretain long-term efficacy with chronic administration.

Note— RTB is one of a class of lectin that function similarly althoughthe sugar binding specificity may differ among lectins.

MPS I is one example of many therapies that could benefit fromtechnology

EXAMPLE 5

Lectin-mediated delivery of ERT and lysosomal disease correction occursin animals previously immune-sensitized to the ERT drug. The potentialof our lectin-based carrier to effectively deliver corrective enzyme istested in MPS I/Hurler mice that are immune-sensitized to the rhIDUdrug. 1) MPS I (idua^(−/−)) mice are immunized with rhIDU to yieldanimals with high-titer neutralizing anti-rhIDU antibodies). 2) Bothsensitized and naïve mice are provided weekly enzyme treatments ofeither rhIDU or IDUA:RTB and impacts on IDUA activity and GAG levels inselective organs and fluids is compared (FIG. 9 ).

A. Develop MPS I mice that are immune-sensitized to the rhIDU ERTproduct.

Rationale. Our first goal is to establish the immunization protocol toproduce idua^(−/−) mice with high-titer anti-rhIDU antibodies. Twostrategies have previously been used to develop immune-sensitizedidua^(−/−) animals: 1) weekly rhIDU administrations at the human dose(0.58 mg rhIDU/Kg) or higher, reflecting patient treatment protocols¹,or 2) adjuvanted immunizations with ERT, patterned after vaccinationprotocols (Turner et al., 2000; Clements et al., 1985; Ashton et al.,1992). The latter approach requires less enzyme, fewer administrations,and provides a greater proportion of high-titer animals (100% (Turner etal., 2000)) compared to non-adjuvanted rhIDU alone and will thus be usedfor these studies. We will test 2 rhIDU doses, 1 μg and 5 μg/mouse,administered with Freund's complete (prime) or Freund's incomplete (3boosts) adjuvant. The 5 μg/mouse dose matches immunization protocolsreported in rats (50 μg rhIDU dose equivalent to ˜0.2 mg/kg) whichyielded 100% high-titer animals (Turner et al., 2000).

The MPS 1 mice effectively model human Hurler syndrome with similarbehavioral disease development, lack of IDUA catalytic activity,considerable GAG accu internal organs, and 3-fold higher urinary GAGlevels than normal mice (Wang et al., 2010; Ou et al., 2014). IDUA-crossreactive protein is not detectable by westerns (Keeling. UAB, pers.comm.). Serum anti-rhIDU IgG levels is measured by ELISAs beforeimmunization and in samples collected following the 2^(nd) and 3^(rd)boosts (see FIG. 10 ). The presence of neutralizing antibodies isassessed based on serum-mediated inhibition of enzyme uptake into humanMPS I fibroblasts (see FIG. 1 ). After the final boost, urine iscollected weekly for three weeks from immunized and age-matchednon-immunized MPS I mice for GAG level determinations (see FIG. 10 ).Urinary GAG levels provide a non-invasive way to assess MPS I diseaseand correction (Dickson et al., 2008). Based on these initial trials ona small cohort of animals, we select a dose and boost schedule for alarger cohort to support Aim 2 studies, which compare enzyme treatmentsin immunized and non-immunized animals. For this trial, 18 idua^(−/−)mice (6-8 wks old) are immunized with the selected adjuvanted protocol.Mice are bled 6 days after the final boost and their sera analyzed foranti-rhIDU antibodies and fibroblast uptake neutralization. Confirmedhigh-titer mice are then used in conjunction with non-immunized mice fordisease treatment studies.

Immunizations. For initial immunization protocol assessment, two groupsof idua^(−/−) mice (6-8 weeks; n=4) are immunized subcutaneously witheither 1 μg or 5 μg rhIDU (assuming 25 g mouse at either 0.04 or 0.2mg/kg dose). Initial immunization is adjuvanted 1:1 with Freund'scomplete adjuvant with boosts at 2-week intervals using enzyme plusFreund's incomplete adjuvant (administration volumes of 100-150 μl).Animals are bled (orbital or tail vein) prior to first immunization and6 days after 2^(nd) and 3^(rd) boosts. A larger cohort (n=18) issubsequently immunized at a single selected rhIDU dose and boostingschedule to support Aim 2 studies. These animals are bled 6 days afterfinal boost.

Assessment of anti-rhIDU serum titers. Serum titers are analyzed byELISA as described (Kakkis et al., 2004). Briefly, 96-well plates arecoated with rhIDU protein (200 ng/well), washed, and incubated with aserum dilution series. Bound antibodies are detected with AP-conjugatedrabbit anti-mouse-IgG antibodies (absorbance 405 nm). Data are presentedbased on OD units/ml serum based on dilutions read within the linearrange. High-titer animals are defined as those having OD units greaterthan 5 OD units/ml serum.

Uptake inhibition assays in MPS I fibroblasts. To determine ifhigh-titer serum from sensitized animals contains antibodies that blockM6P-mediated uptake, an antibody-mediated uptake inhibition assay isperformed as described (Dickson et al., 2008) (see also FIG. 1 thattested uptake in presence of canine serum). rhIDU is pre-incubated inmedia with mouse serum for 1 hour prior to addition to cells. Severalserum dilutions are tested (1:1000, 1:500); for canine serum, 1:1000dilution provided >90% inhibition of rhIDU uptake¹ (FIG. 1 ). Afterincubation with confluent cultures, cells are harvested, andintracellular IDUA activity is measured in cell lysates using standardfluorometric assays with 4-MU-iduronide. Percentage of uptake inhibitionis calculated by comparing intracellular IDUA activity of cellsincubated with rhIDU+/− serum.

Measurement of urinary GAG. Urine samples are collected from individualmice over a 24 hr period (e.g., using metabolic cages), sterilefiltered, and stored at 4° C. until assayed. GAG content is quantifiedusing the dimethylmethylene blue chloride (DMMB) as described (Wang etal., 2010; De Jong et al., 1992). GAG levels are normalized tocreatinine and expressed as mg GAG per mg creatinine (Wang et al., 2010;De Jong et al., 1992).

These studies establish specific immunization parameters (rhIDUimmunogen dose, number of boosts) required to yield stronguptake-blocking immune response in MPS I mice. These conditions are thenused to produce a cohort of rhIDU-sensitized mice for disease treatment.

B. Compare IDUA Activity and GAG Levels in Selected Tissues inrhIDU-Sensitized Mice Following Short-Term Treatments with EitherCommercial rhIDU or IDUA:RTB

Rationale. The hypothesis that IDUA:RTB can deliver corrective doses ofIDUA enzyme in animals with high-titer anti-rhIDU antibodies is tested.The overall strategy is summarized in FIG. 9 . The immunization phase(described above) produces rhIDU-immunized MPS I mice with immunesensitization. status qualified by serum anti-rhIDU antibody levels andcell uptake inhibition assays. In the treatment phase, confirmedhigh-titer immunized and age-matched non-immunized. MPS I mice aretreated intravenously weekly for a total of 4 treatments with rhIDU orplant-made IDUA:RTB (see Table 5). Each treatment provides the humantherapeutic dose equivalent of 0.58 mg IDUA/kg. E.g., for a 25 g mouse,this are 14.5 μg rhIDU (˜85 kDa) or 20.5 μg IDUA:RTB (˜120 kDa), IDUAenzyme activity and GAG levels are assessed in heart and kidney 4 daysafter final therapeutic treatment.

TABLE 5 Animal treatments. Animal Number Immunization ERT Treatment MPSI (idua^(−/−)) n = 3-6* rhIDU-immunized rhIDU MPS I (idua^(−/−)) n = 3None rhIDU MPS I (idua^(−/−)) n = 3-6* rhIDU-immunized IDUA:RTB MPS I(idua^(−/−)) n = 3 None IDUA:RTB MPS I (idua^(−/−)) n = 3-6*rhIDU-immunized None MPS I (idua^(−/−)) n = 2 None None Normal(idua^(+/+)) n = 2 None None *6 mice will be immunized; at least 3 willbe treated and/or analyzed

Heart and kidney are selected as the primary organs for these analysesbased on the following: In multiple studies assessing impacts ofanti-drug antibodies on enzyme therapy or testing potential tolerizationstrategies, alterations in therapeutic enzyme bio-distribution providedthe most reliable short-term indicator of immune-sensitization (Dicksonet al., 2008; Glaros et al., 2002). However, impacts on specific organsdiffer—organs such as liver that are rich in macrophages andreticuloendothelial cells may actually have elevated IDUA levels inhigh-titer individuals, putatively linked with antibody-directed (asopposed to M6P—directed) uptake. In contrast, kidney, heart and lungconsistently show reduced IDUA activity and higher GAG levels inrhIDU-sensitized animals compared to low-titer (non-immunized orimmune-tolerized) animals (Dickson et al., 2008; Glaros et al., 2002).In recent experiments, the. Dickson group demonstrated that heart andkidney rhIDU activity levels were reduced by more than 50% in high-titer(3-30 OD units/ml) MPS I mice compared to low-titer (<1 OD unit/ml) micefollowing 4 weekly doses (Dickson, pers. comm.; manuscript submitted).Heart and kidney produced the most dramatic and statisticallysignificant differences in high-versus low-titer animals. Therefore,initial analyses are restricted to these organs. Follow-on studiesprovide more thorough investigations of IDUA:RTB biodistribution andefficacy in immune-sensitized mice.

The selection of heart and kidney for the analyses is also supported byour in viva studies with IDUA:RTB. Initial trials with IDUA:RTBadministered to MPS I mice demonstrate that our plant-made product iseffectively taken up by heart and kidney, as well as other organs(Acosta et al., 2016; Ou et al., 2016). As shown in FIG. 1.1A,substantial. IDUA activity was detected in these organs 24 hr afteradministration (levels in kidney were equivalent WT). Because this wasthe first in vivo administration of IDUA:RTB, we also treated 3 micewith a 10× dose (5.8 mug IDUA equivalents/kg) and monitored them for 5days for any adverse effects. No signs of toxicity were observed and, atendpoint, these mice were analyzed for tissue GAG levels. SignificantGAG clearance was seen in kidney and heart (FIG. 11B) as well as liverand spleen (data not shown). These results provide confidence that IDUAactivity and IDUA:RTB-mediated GAG reduction are detectable in heart andkidney tissue in non-immune MPS I mice and that the experimental designshould be effective in demonstrating that IDUA:RTB, but not rhIDU, canmediate GAG reduction in the presence of high antibody titers.

Production of plant-derived IDUA:RTB. At BioStrategies, bioproductionand purification protocols for IDUA:RTB has been established to generatedefined, well characterized protein to support mouse biodistribution andefficacy studies. Transient expression in Nicotiana benthamiana (Whaleyet al., 2011; Komarova et al., 2010) is accomplished by vacuuminfiltration of intact plants with Agrobacterium tumefaciens carryingthe IDUA:RTB construct. After four days, leaves are harvested and asimple 3-step purification protocol yields product with >95% purity.Established quality control protocols for the final product includequantification of lectin binding activity, IDUA enzyme units, proteinconcentration by absorbance at 280 nm, and endotoxin levels using amodified Limulus Amebocyte Lysate (LAL) assay (detects to 0.005-1EU/ml).

Enzyme replacement treatments. High-titer immunized mice (see above) andage-matched naïve mice are administered IDUA enzyme (0.58 mg IDUAequivalent/kg in PBS (<150 μl) rhIDU or IDUA:RTB by tail vein injection)weekly starting two weeks after the immunized group receives the finalboost. Mice are monitored carefully for injection-related stress. After4 treatments, mice are euthanized, perfused, and selected organsisolated, weighted and snap-frozen. Various control groups (see Table 5)are processed in parallel. Analyses of heart and kidney IDUA activityand GAG levels in tissue homogenates are described previously (Wang etal., 2010; Ou et al., 2014).

Statistical analyses. Evaluation of differences between samples areanalyzed using Tukey test for comparisons between paired samples and twoway analysis of variance (ANOVA) for comparisons between three or moresamples. Statistical significant level are set at p<0.05. This studyrepresents a preliminary proof-of-concept feasibility test. Moreextensive preclinical assessments will further support power statisticalanalyses.

These studies compare IDUA:RTB and rhIDU enzyme treatments onrhIDU-immunized and non-immunized MPS I mice and analyze impacts on IDUAactivity and GAG levels in kidney and heart (e.g., FIGS. 11A and 11B).These represent technically straight-forward procedures for which theprotocols and expertise are established and the strategy iswell-supported by the literature. These studies show IDUA:RTB is able tocircumvent high anti-rhIDU serum titers and successfully delivercorrective enzyme in high-titer mice. The definitive data are thecomparison of heart/kidney IDUA activity and GAG levels in immunizedmice that are treated with IDUA:RTB versus rhIDU. IDUA:RTB treated miceshow 1) higher IDUA activity and significant GAG correction (e.g. 50% ofuntreated MPS I mice) and 2) greater GAG reduction in heart and kidneythan rhIDU-treated animals. Based on previous studies, including our ownpreliminary data with IDUA:RTB, 4 weekly treatments at the proposed ERTdose provided sufficient differences in treated versus untreated GAGlevels to distinguish ERT efficacy among the groups (Dickson et al.,2008; Ou et al., 2014). We have included various control groups(untreated MPS I mice, immunized/untreated MPS I mice,non-immunized/treated mice) to 1) document that both the rhIDU andIDUA:RTB enzymes are functional and provide the expected treatmentoutcomes in non-immunized mice and 2) provide baseline “normal” andbaseline “MPS I disease” levels from age-matched siblings. Boththerapeutic enzymes are qualified (quantity, purity, IDUA activity)before administration, but experiments are repeated with new qualifiedenzyme or higher ERT doses are tested if control treatments (naïve MPS Imice) fail to show the expected reduction in heart/kidney GAG levels.

These studies delineate the novel delivery mechanisms of IDUA:RTB tocircumvent the inhibition of therapeutic efficacy imposed by circulatinganti-rhIDU neutralizing antibodies. Additional studies assess: IDUA:RTBbiodistribution and pharmacodynamics in immunized and non-immunizedanimals; IDUA:RTB immunogenicity; proof of concept in other LSD diseasesincluding Pompe, GM1 gangliosidosis, Hunter and other diseases andtreatments where the prevalence of immune responses is underminingefforts to bring new ERTs or gene therapy options to patients (Wang etal., 2008; Kishnani et al., 2010; Xu et al., 2004).

EXAMPLE 6

Example 5 highlights utility for the lectincarrier—therapeutic/bioactive molecule fusion to effectively treatindividuals that have previously developed ADA to the therapeutic entitythat undermines treatment efficacy. Example 6 provides additionaladvantages of the technology in treatment of “naïve” individuals suchthat even if they develop ADA antibodies during chronic treatment, thecarrier continues to delivery to disease-critical cells and tissues andthe individual avoids ADA-mediated decline in treatment efficacy.

Disease knockout mice, such as the MPS I or Pompe mouse models, aretreated with a normal or 10× normal corrective doses (e.g. 0.58 mg/kgand 5.8 mg/kg IDUA equivalents for MPS I mice) at weekly intervals usingeither mammalian cell-derived enzyme (e.g., rhIDU) or lectin-carrierfused enzyme (e.g., IDUA:RTB). Urine GAG levels (or equivalent readoutsassociated with the specific disease) are assessed weekly and serumantibodies assessed every 3 weeks. The number of animals that showimprovement (e.g., low urine GAG) followed by decline (e.g., elevatedurine GAG) correlated with anti-enzyme antibody titers are comparedamong treatment groups. At the treatment stage showing a significantdifference between the enzyme vs. the enzyme:RTB treatment, selectedanimals will be sacrificed to assess liver, kidney, spleen and heartenzyme and GAG levels. The utility of RTB as a long-term carrier isexemplified by continuous treatment efficacy that does not show theADA-associated decline in treatment efficacy.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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We claim:
 1. A method for delivering an agent into a cell, in thepresence of immune components against said agent, the method comprisingproviding said agent in an environment comprising said cell andcomprising said immune components directed against said agent, whereinsaid immune components are immune cells or neutralizing antibodies orantisera that bind to one or more epitopes of said agent, and whereinsaid agent is operatively linked to a lectin-based carrier, and wherebysaid agent operatively linked to said lectin-based carrier isinternalized into said cell by uptake mechanisms directed by thelectin-based carrier.
 2. The method according to claim 1, wherein saidlectin-based carrier is a plant lectin, or wherein said lectin-basedcarrier is a non-toxic carbohydrate binding subunit of a plant toxin. 3.The method according to claim 1, wherein said lectin-based carrier is oflow immunogenicity or non-immunogenic.
 4. The method according to claim1, wherein said lectin-based carrier is the B-subunit of ricin.
 5. Themethod according to claim 1, wherein said agent is a compound, drug,protein, peptide, antigen, immunogen, or nucleic acid, or wherein saidagent is a lysosomal enzyme.
 6. The method according to claim 1, whereinsaid cell is a mammalian cell.
 7. A method for treating a disease orcondition in a human or animal that has previously developed an immuneresponse against a therapeutic agent that can treat said disease orcondition, wherein said immune response comprises the production ofimmune cells or neutralizing antibodies that bind to one or moreepitopes of said therapeutic agent, the method comprising administeringto the human or animal an effective amount of said therapeutic agentoperatively linked to a lectin-based carrier.
 8. The method according toclaim 7, wherein said lectin-based carrier is a plant lectin, or whereinsaid lectin-based carrier is a non-toxic carbohydrate binding subunit ofa plant toxin.
 9. The method according to claim 7, wherein saidlectin-based carrier is the B-subunit of ricin.
 10. The method accordingto claim 7, wherein said lectin-based carrier is of low immunogenicityor non-immunogenic.
 11. The method according to claim 7, wherein saidagent is a compound, drug, protein, peptide, antigen, immunogen, nucleicacid, or other synthetic or biological molecule that is used in treatingor preventing the disease or condition, or wherein said agent is alysosomal enzyme.
 12. The method according to claim 7, wherein saiddisease or condition is a lysosomal storage disease, or wherein saiddisease or condition is Hurler disease.
 13. The method according toclaim 1, wherein said cell has a lysosomal storage disorder, or whereinsaid cell is deficient in production of α-L-iduronidase.
 14. The methodaccording to claim 5, wherein said lysosomal enzyme is iduronidase. 15.The method according to claim 11, wherein said lysosomal enzyme isiduronidase.
 16. The method according to claim 14, wherein saidlectin-based carrier is the B-subunit of ricin.
 17. The method accordingto claim 15, wherein said lectin-based carrier is the B-subunit ofricin.